ErbB/HER receptors play essential roles in propagating signals that regulate cell proliferation, differentiation, motility, and apoptosis. Significantly, aberrant regulation of the ErbB/HER family of receptor tyrosine kinases and their ligands is a common occurrence in many human tumors. Indeed, EGFR, HER2, and HER3 have been diagnostically and therapeutically validated as targets in a wide variety of human malignancies such as lung, colorectal, breast, head and neck, gastric and pancreatic cancers. Therefore, novel protein biopharmaceuticals that comprehensively target ErbB/HER family members could have significant applications in targeted diagnosis and therapy of different types of cancer. This project addresses a critical need for developing cost-effective targeting ligands that recognize and bind HER family members with desired avidity and multispecificity. The project is directed at using a powerful in vitro protein selection method called mRNA display to evolve single domain antibodies (SDAs) with human origin that tightly and specifically bind to the extracellular domain of EFRG, HER2 or HER3, respectively, from a human SDA domain library with an unusually high diversity. The resulting monomeric SDAs will be used as the basis for the generation of heterodimeric SDAs that bind to a HER member of interest at two nonoverlapping epitopes with synergistic avidity effect. In addition, we will develop an efficient and universal self-assembly system that allows for reversible and controllable loading of HER-binding SDAs to nanoparticles to acquire desired multispecificity. The resulting SDAs would have a number of advantages, including high target-binding affinity, ease to achieve desired avidity, multivalency and multispecificity, minimal immunogenicity due to human origin, small size for better tissue penetration, in addition to significantly reduced manufacturing costs due to high expression levels in bacteria. The simplicity and stability of these SDAs also greatly facilitate their conjugation with various nanoparticles for translational applications. The resulting SDAs will be examined in vivo in pancreatic ductal adenocarcinoma (PDAC) xenograft animal models by targeted delivery of nanoparticles containing siRNAs against several metastasis signature genes that are important for PDAC tumorigenesis and metastasis. The success of the project will lead to the development of cost-effective, targeted biopharmaceuticals for cancer diagnosis and therapy.